WO2010013601A1 - Microneedle device and method for increasing the response of japanese encephalitis virus antigen with the microneedle device - Google Patents

Microneedle device and method for increasing the response of japanese encephalitis virus antigen with the microneedle device Download PDF

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Publication number
WO2010013601A1
WO2010013601A1 PCT/JP2009/062887 JP2009062887W WO2010013601A1 WO 2010013601 A1 WO2010013601 A1 WO 2010013601A1 JP 2009062887 W JP2009062887 W JP 2009062887W WO 2010013601 A1 WO2010013601 A1 WO 2010013601A1
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Prior art keywords
encephalitis virus
japanese encephalitis
microneedle
virus antigen
microneedle device
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PCT/JP2009/062887
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French (fr)
Japanese (ja)
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野崎周英
上仲一義
松田純一
寺原孝明
桑原哲治
徳本誠治
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久光製薬株式会社
財団法人化学及血清療法研究所
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Priority to JP2010522676A priority Critical patent/JP5744517B2/en
Priority to CN2009801302909A priority patent/CN102112151A/en
Publication of WO2010013601A1 publication Critical patent/WO2010013601A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a method for enhancing immunogenicity using a microneedle device.
  • the skin consists of the outermost stratum corneum, epidermis, dermis, and subcutaneous connective tissue.
  • stratum corneum consisting of a dead cell layer and a lipid bilayer exhibits a strong barrier function against many substances.
  • antigen-presenting cells called Langerhans cells exist and have an immune function. Langerhans cells capture protein antigens that have entered the skin, break down inside, and express peptide fragments on MHC molecules.
  • the MHC-peptide complex moves from the imported lymphatic vessel to the subcortical layer of the regional lymph node, and contacts through T cells and finger process cells. Langerhans cells, by moving in this manner, is transmitted to the T H cells antigen is present in the lymph nodes from the skin. Langerhans cells have MHC class II molecules necessary to present antigen to the T H cells.
  • the virus antigen to the dermis layer is effective due to the strong barrier function by the stratum corneum of the skin, but the administration by the injection needle to the dermis layer limited to 300 to 2000 ⁇ m is technical. There was a problem in accuracy due to difficulty.
  • a microneedle has been developed as a means for solving this problem. These are intended to puncture the outermost stratum corneum, and have been devised in various sizes and shapes (very small protrusions with a height of several tens to several hundreds of micrometers), especially non-invasive It is expected as a new virus antigen administration method.
  • microneedles various methods have been devised for applying drugs when using a device equipped with microneedles, such as a method of coating a drug on the surface of the microneedle and administering the drug or a biological component through the needle.
  • a method of forming a hole (hollow needle) or a groove for making the needle, a method of mixing a drug in the needle itself, and the like have been proposed.
  • Each of these microneedle devices has very small protrusions (microneedles) with a height of several tens to several hundreds of micrometers, so depending on the method of applying the drug, Absorption efficiency is also considered to differ greatly.
  • Non-Patent Document 1 As a method for efficiently promoting transdermal absorbability of an antigen (vaccine) using a microneedle, there is a method of coating a drug on a part of the surface of the microneedle, which is disclosed in Non-Patent Document 1, for example. This is useful when a part of the microneedle (especially only the needle part) is coated with an antigen (vaccine), or all or most of the applied antigen (vaccine) is transferred into the body and is useful as an accurate dermis administration means. It shows that there is.
  • Non-patent Document 2 Japanese encephalitis virus antigens
  • Patent Document 1 discloses administration with a microneedle having a hollow needle for the purpose of reducing the amount of a therapeutic substance and achieving a therapeutic effect, but does not describe immunity induction.
  • An object of the present invention is to provide a method for enhancing immunogenicity using a microneedle device that enhances the immunogenicity of a Japanese encephalitis virus antigen.
  • the present inventor has conducted daily studies with the above as a technical background, and as a result, transdermally administers Japanese encephalitis virus antigens using microneedles coated with Japanese encephalitis virus antigens composed of antigens derived from monkey kidney cells (Vero cells). And found that the antibody property increased. Furthermore, after the transdermal administration of microneedles coated with Japanese encephalitis virus antigen, a patch containing lauryl alcohol was affixed, whereby a further efficient increase in antibody titer could be confirmed.
  • the method for enhancing immunogenicity using a microneedle device is a method for treating a microneedle device comprising a microneedle made of polylactic acid coated with a Japanese encephalitis virus antigen made of an antigen derived from monkey kidney cells (Vero cells). And the Japanese encephalitis virus antigen is transdermally administered.
  • the immunogenicity is further enhanced by applying a patch containing lauryl alcohol having an adjuvant effect.
  • the application of a substance having an adjuvant activity that can penetrate the skin can be expected to enhance the immunogenicity.
  • the microneedle device includes a microneedle composed of polylactic acid coated with a Japanese encephalitis virus antigen composed of an antigen derived from monkey kidney cells (Vero cells).
  • the coating preferably includes pullulan as a coating carrier.
  • the immunogenicity can be increased by a simpler operation than injection of Japanese encephalitis virus antigen consisting of monkey kidney cell (Vero cell) -derived antigen.
  • Vero cell monkey kidney cell
  • the immune response induced by Japanese encephalitis virus antigens can be stimulated, and the effective dose of antigens in the virus antigens can be reduced.
  • FIG. 1 is a view showing an example of a microneedle device according to the present invention, where (a) is a perspective view and (b) is a cross-sectional view taken along line AB of (a).
  • a microneedle device (interface) 5 according to the present invention comprises a microneedle substrate 8 and a plurality of microneedles 6 arranged in a two-dimensional shape capable of perforating the skin or mucous membrane.
  • the microneedle substrate 8 includes a plurality of openings 7 arranged corresponding to the microneedles 6.
  • the shape of the microneedle 6 is a conical shape, but the present invention is not limited to this, and may be a polygonal pyramid such as a quadrangular pyramid or another shape.
  • the plurality of microneedles 6 and the plurality of openings 7 are alternately arranged in a square lattice shape, the present invention is not limited to this.
  • the number of the microneedles 6 and the openings 7 is 1: 1 in the drawing, the present invention is not limited to this, and includes those that do not include the openings 7.
  • a part or the whole of the microneedle 6 is coated with a Japanese encephalitis virus antigen composed of an antigen derived from monkey kidney cells (Vero cells).
  • the coating 1 is arrange
  • the coating 1 is disposed on the entire microneedle 6, but may be disposed on a part thereof.
  • the microneedle substrate surface on which the microneedles 6 shown in FIG. 1 (a) are disposed is brought into contact with the skin and a drug solution is allowed to flow from the opposite surface, the liquid flows out from each opening 7 and each micro It is transmitted to the needle 6 and the Japanese encephalitis virus antigen is percutaneously absorbed.
  • the opening 7 is not essential, and the liquid may be supplied to the microneedle 6 by another means without using the opening 7.
  • the coating 1 can release the Japanese encephalitis virus antigen into the skin by dissolving with a body fluid when the microneedle is perforated in the skin without applying a liquid from the outside.
  • the microneedle in the microneedle device includes a microneedle (needle) that is punctured into the skin or mucous membrane and a substrate that supports the needle portion, and a plurality of microneedles are arranged on the substrate.
  • the microneedle has a microstructure, and the height (length) h of the microneedle is preferably 50 ⁇ m to 700 ⁇ m, more preferably 100 ⁇ m to 600 ⁇ m, and further preferably 200 ⁇ m to 500 ⁇ m.
  • the length of the microneedle is set to 50 ⁇ m or more to ensure administration of the Japanese encephalitis virus antigen from the skin, and the length of 700 ⁇ m or less avoids contact of the microneedle with the nerve, This is because the possibility of pain can be surely reduced and at the same time the possibility of bleeding can be surely avoided. If the length is 700 ⁇ m or less, the amount of Japanese encephalitis virus antigen that enters the skin can be efficiently administered.
  • the microneedle means a convex structure and a needle shape in a broad sense or a structure including a needle shape.
  • the diameter at the base is usually about 50 to 200 ⁇ m.
  • the microneedle is not limited to a needle shape having a sharp tip, and includes a shape having no sharp tip.
  • the microneedles are preferably made using a non-metallic synthetic or natural resin material.
  • the shape of the microneedle is a conical shape in this example, but the present invention is not limited to this, and may be a polygonal pyramid such as a quadrangular pyramid or another shape.
  • the microneedle substrate is a base for supporting the microneedle, and the form thereof is not limited.
  • the substrate may be a substrate having a through hole (opening) as shown in FIG.
  • the Japanese encephalitis virus antigen can also be administered by flowing through the opening and the microneedle.
  • the material of the microneedle or the substrate include silicon, silicon dioxide, ceramic, metal (stainless steel, titanium, nickel, molybdenum, chromium, cobalt, etc.) and synthetic or natural resin materials.
  • biodegradable polymers such as polylactic acid, polyglycolide, polylactic acid-co-polyglycolide, pullulan, capronolactone, polyurethane, polyanhydride, polycarbonates that are non-degradable polymers, polymethacrylic acid,
  • a synthetic or natural resin material such as ethylene vinyl acetate, polytetrafluoroethylene, or polyoxymethylene is particularly preferable.
  • polysaccharides such as hyaluronic acid, pullulan, dextran, dextrin or chondroitin sulfate.
  • polylactic acid is a biodegradable resin and has been used as an implant preparation (Japanese Patent Publication No. 2002-517300 or Journal of Controlled Release 104 (2005) 51-66). It is one of the most suitable microneedle materials in view of the above.
  • the density of microneedles is typically spaced between the rows such that the rows of needles provide a density of about 1 to 10 per millimeter (mm).
  • the rows are separated by a substantially equal distance from the needle space in the row and have a needle density of 100-10000 per cm 2 , preferably 100-5000, more preferably 200-2000, and even more The number is preferably 400 to 1000.
  • the needle density exceeds 10,000, it is difficult to give the microneedles strength capable of perforating the skin.
  • Microneedle manufacturing methods include wet etching or dry etching using a silicon substrate, precision machining using metal or resin (electric discharge machining, laser machining, dicing, hot embossing, injection molding, etc.), machinery Examples include cutting.
  • the needle portion and the support portion are integrally molded.
  • As a method for making the needle part hollow there is a method of performing secondary processing by laser processing or the like after producing the needle part.
  • the temperature and humidity of the installation environment of the apparatus can be controlled to be constant in order to minimize changes in drug concentration and physical properties due to solvent volatilization of the coating solution.
  • the humidity at room temperature is 50 to 100% RH as relative humidity, and preferably 70.0 to 100% RH. If it is 50% RH or less, the solvent may evaporate and the physical properties of the coating solution may change.
  • the humidification method is not particularly limited as long as the target humidity state can be secured, but there are a vaporization method, a steam method, a water spray method, and the like.
  • Preparation of the Japanese encephalitis virus antigen used in the present invention can be performed with reference to, for example, WO01 / 076664.
  • the coating liquid for coating the microneedles can contain a coating carrier and a liquid composition in addition to the Japanese encephalitis virus antigen.
  • the coating of the present invention is preferably a state in which the coating liquid stays and adheres to the microneedles (needles).
  • the coating liquid may be fixed by applying a drying step.
  • a polysaccharide carrier that is relatively compatible with the Japanese encephalitis virus antigen (a property that uniformly crosses) is preferable.
  • Polyhydroxymethylcellulose, hydroxypropylcellulose, polyhydroxypropylmethylcellulose, polymethylcellulose, dextran, polyethylene glycol, Pullulan, carmellose sodium, chondroitin sulfate, hyaluronic acid, dextran, gum arabic and the like are preferred, and hydroxypropyl cellulose, pullulan and gum arabic are more preferred.
  • HPC-SSL molecular weight: 15,000 to 30,000
  • HPC-SL molecular weight: 30,000 to 50,000
  • HPC-L molecular weight: 55,000 to 70,000
  • HPC-M molecular weight: 110,000 to 150,000
  • HPC-H molecular weight: 250,000 to 400,000
  • pullulan is most preferable in terms of compatibility with the Japanese encephalitis virus antigen.
  • the content of the coating carrier in the entire coating liquid is 1 to 70% by weight, preferably 1 to 40% by weight, particularly preferably 3 to 25% by weight.
  • this coating carrier may require a certain degree of viscosity so that it does not drip, and the viscosity is required to be about 100 to 100,000 cps. A more preferred viscosity is 500 to 60000 cps. When the viscosity is in this range, it is possible to apply a desired amount of the coating liquid at a time without depending on the material of the microneedle. In general, the higher the viscosity, the greater the amount of coating solution.
  • the liquid composition used to coat the microneedles is prepared by mixing a biocompatible carrier, the Japanese encephalitis virus antigen to be delivered, and possibly any coating aids with a volatile liquid.
  • the volatile liquid can be water, dimethyl sulfoxide, dimethylformamide, ethanol, isopropyl alcohol, mixtures thereof, and the like. Of these, water is most preferred.
  • the liquid coating solution or suspension can typically have a Japanese encephalitis virus antigen concentration of 0.1 to 65% by weight, preferably 1 to 30% by weight, more preferably 3 to 20% by weight. %.
  • Other known formulation adjuvants may be added to the coating as long as they do not adversely affect the required solubility and viscosity characteristics of the coating and the properties and physical properties of the dried coating.
  • the thickness of the microneedle coating is less than 50 ⁇ m, preferably less than 25 ⁇ m, more preferably 1 to 10 ⁇ m. In general, the coating thickness is the average thickness measured across the surface of the microneedle after drying.
  • the thickness of the coating can generally be increased by applying multiple coatings of the coating carrier, i.e. by repeating the coating process after fixing the coating carrier.
  • the height (length) h of the microneedle is preferably 50 ⁇ m to 700 ⁇ m as described above.
  • the coating height H of the microneedle varies depending on the height h of the microneedle, but can be in the range of 0 ⁇ m to 700 ⁇ m, usually in the range of 10 ⁇ m to 700 ⁇ m, and preferably about 30 ⁇ m to 500 ⁇ m. is there.
  • an aliphatic alcohol having an adjuvant effect can be applied to the site administered by the microneedle device of the present invention.
  • these aliphatic alcohols linear or branched aliphatic alcohols are preferred.
  • the carbon number and molecular weight are not particularly limited, but considering the skin permeability, it is more preferably 8 to 20 carbon atoms.
  • Such aliphatic alcohols may be saturated or unsaturated.
  • these fatty alcohols they are often used as absorption enhancers in percutaneous absorption.
  • the aliphatic alcohols in the present invention have an adjuvant effect in addition to the absorption promoting action. You can expect.
  • Such aliphatic alcohols are, for example, octyldodecanol, lauryl alcohol, oleyl alcohol, isostearyl alcohol, decanol, etc.
  • lauryl alcohol, oleyl alcohol, and isostearyl alcohol are particularly preferable, and lauryl alcohol is most preferable. .
  • the aliphatic alcohol of the present invention is preferably blended at 0.1 to 99% by weight, more preferably 5 to 90% by weight, particularly 10 to 80% by weight. More preferably.
  • the content of aliphatic alcohols in the most preferred composition is 15 to 75% by weight.
  • the form of the pharmaceutical preparation containing the adjuvant is not particularly limited as long as it can be administered transdermally. However, patches, ointments, creams, solutions, gels, lotions, etc. However, a patch preparation is particularly preferable.
  • the administration site of the patch preparation is not particularly limited, but is preferably closer to the antigen administration site, and more preferably applied to the upper part of the antigen administration site.
  • the preparation for transdermal administration contains, as a base, optional components such as a solubilizer, a solubilizer, a pH adjuster, an antiseptic, an absorption accelerator, a stabilizer, a filler, a thickener, an adhesive, and a wetting agent.
  • composition of the pharmaceutical preparation containing the adjuvant is preferably 0.1 to 99% by weight, more preferably 5 to 90% by weight, and even more preferably 10 to 80% by weight.
  • content of aliphatic alcohols in the most preferred composition is 15 to 75% by weight.
  • Example 1 Japanese encephalitis virus antigens containing antigens derived from monkey kidney cells (Vero cells) prepared as follows were concentrated by centrifugation using BIOMAX-10K (Millipore), mixed with a polymer (pullulan), While maintaining a relative humidity of 90 to 100% HR with a humidifier, the microneedle made of polylactic acid (height of about 300 ⁇ m, density of 841 pieces / cm 2 , square pyramid shape) was coated. The coating content was 2 ⁇ g / patch of antigen, and a 4-week-old ddY mouse (female) was shaved from the abdomen under anesthesia, and then the coated microneedle was punctured for 2 hours into the skin (4 cases).
  • an adjuvant (lauryl alcohol) patch was affixed to the upper part of the microneedle administration site.
  • boosting was performed under the same conditions.
  • the Japanese encephalitis virus antigen-specific IgG antibody titer was measured. The results are shown in FIG.
  • the collected blood was allowed to stand overnight at 4 ° C., and then the serum was separated by centrifugation and inactivated (treated at 56 ° C. for 30 minutes).
  • the virus antigen diluted to 5 ⁇ g / mL with the coating buffer was added to the well at 100 ⁇ L / well and allowed to stand at 4 ° C. overnight.
  • the solid-phased plate was washed 3 times with 400 ⁇ L / well of washing buffer, 250 ⁇ L / well of blocking buffer was added, and reacted at 37 ° C. for 1 hour.
  • the sample was washed 3 times with a washing buffer of 400 ⁇ L / well, sufficiently drained, then diluted 100-fold with a dilution buffer, and 100 ⁇ L / well of each sample diluted serially in a 2-fold series was added at 37 ° C. for 2 hours. Reacted.
  • the plate was washed 3 times with a washing buffer of 400 ⁇ L / well, diluted HRP-labeled antibody 100 ⁇ L / well was added, and reacted at 37 ° C. for 90 minutes.
  • a sufficient Japanese encephalitis virus antigen-specific IgG antibody titer can be obtained with the microneedle device (MN) alone, and even higher when the adjuvant (lauryl alcohol) patch is used together (MN + LAtape). Encephalitis virus antigen-specific IgG antibody titers can be obtained.
  • the present invention includes the following. (1) A plurality of microneedles that are two-dimensionally arranged on a substrate and are made of polylactic acid capable of perforating the skin, and coated with Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells). Microneedle device. (2) The microneedle device according to (1) above, wherein the microneedle is conical or pyramidal. (3) The microneedle device according to (1) or (2), wherein the coating contains pullulan as a coating carrier. (4) The microneedle device according to any one of (1) to (3), wherein the coating is performed at a relative humidity of 70.0 to 100% RH at room temperature.
  • microneedle device (5) The microneedle device according to any one of (1) to (4), wherein the coating contains a substance having an adjuvant activity.
  • the substance having adjuvant activity is lauryl alcohol.
  • Microneedle device (8) The microneedle device according to any one of (1) to (7), wherein the height of the microneedle is 200 to 500 ⁇ m.
  • microneedle device according to any one of (1) to (8), wherein the microneedles are arranged at a density of 400 to 1000 needles / cm 2 .
  • a method for increasing the response of Japanese encephalitis virus antigen by administering a patch containing a substance having an adjuvant activity after administration of Japanese encephalitis virus antigen using a microneedle device.
  • (13) The method for increasing the response of Japanese encephalitis virus antigen by the microneedle device according to (11) or (12), wherein the coating contains pullulan as a coating carrier.
  • a patch having further adjuvant activity after transdermal administration of Japanese encephalitis virus antigen by the method for increasing the efficacy of Japanese encephalitis virus antigen by the microneedle device according to any of (11) to (19) above A method of increasing the response of Japanese encephalitis virus antigens with a further microneedle device.
  • the present invention makes it possible to increase the antigenicity of a Japanese encephalitis virus antigen comprising a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells) by an efficient and simple operation by using a microneedle device. And has industrial applicability.

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Abstract

Provided is a method for enhancing immunogenicity by using a microneedle device which is capable of enhancing the immunogenicity of a Japanese encephalitis virus antigen. The above-described method for enhancing immunogenicity by using a microneedle device comprises transdermally administering a Japanese encephalitis virus antigen by using a microneedle coated with the Japanese encephalitis virus antigen that is an antigen originating in monkey kidney cells (Vero cells).  Thus, antibody against the Japanese encephalitis virus antigen is effectively elevated.  After transdermally administering the microneedle coated with the Japanese encephalitis virus antigen, a patch containing lauryl alcohol is further applied.

Description

マイクロニードルデバイスおよびマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏功性を上昇させる方法Microneedle device and method for increasing the efficacy of Japanese encephalitis virus antigen by microneedle device
　本発明は、マイクロニードルデバイスによる免疫原性増強方法に関する。 The present invention relates to a method for enhancing immunogenicity using a microneedle device.
　皮膚は、最外層の角質層、表皮、真皮、および皮下結合組織からなる。通常、死細胞層および脂質二重層からなる角質層は、多くの物質に対して強力なバリア機能を示す。真皮層には、ランゲルハンス細胞と呼ばれる抗原提示細胞が存在し免疫機能を担っている。ランゲルハンス細胞は、皮膚内に侵入したタンパク質抗原を補足し、内部で分解し、ＭＨＣ分子上にペプチド断片を表出する。ＭＨＣ－ペプチド複合体は、輸入リンパ管から所属リンパ節の皮質下層へと移動し、Ｔ細胞と指状突起細胞とを介して接触する。ランゲルハンス細胞が、このように移動することによって、抗原が皮膚からリンパ節内に存在するＴＨ細胞へと伝えられる。ランゲルハンス細胞は、抗原をＴＨ細胞へ提示するために必要なＭＨＣクラスＩＩ分子を有している。 The skin consists of the outermost stratum corneum, epidermis, dermis, and subcutaneous connective tissue. Usually, the stratum corneum consisting of a dead cell layer and a lipid bilayer exhibits a strong barrier function against many substances. In the dermis layer, antigen-presenting cells called Langerhans cells exist and have an immune function. Langerhans cells capture protein antigens that have entered the skin, break down inside, and express peptide fragments on MHC molecules. The MHC-peptide complex moves from the imported lymphatic vessel to the subcortical layer of the regional lymph node, and contacts through T cells and finger process cells. Langerhans cells, by moving in this manner, is transmitted to the T H cells antigen is present in the lymph nodes from the skin. Langerhans cells have MHC class II molecules necessary to present antigen to the T H cells.
　このように皮膚の角質層による強力なバリア機能のため真皮層へのウイルス抗原は有効であることは知られていたが、３００～２０００μｍと限られた真皮層への注射針による投与は技術的難しさから精度上の問題があった。 Thus, it was known that the virus antigen to the dermis layer is effective due to the strong barrier function by the stratum corneum of the skin, but the administration by the injection needle to the dermis layer limited to 300 to 2000 μm is technical. There was a problem in accuracy due to difficulty.
　これを解決するための手段として、マイクロニードルが開発されている。これらは、最外層である角質層を穿刺することを目的とし、様々なサイズや形状（高さ数十～数百マイクロメートル程度の非常に小さな突起物）が考案されており、特に非侵襲的なウイルス抗原投与方法として期待されている。 A microneedle has been developed as a means for solving this problem. These are intended to puncture the outermost stratum corneum, and have been devised in various sizes and shapes (very small protrusions with a height of several tens to several hundreds of micrometers), especially non-invasive It is expected as a new virus antigen administration method.
　また、マイクロニードルを備えたデバイスを利用した場合の薬剤の適用方法についても様々な方法が考案されており、薬剤をマイクロニードル表面にコーティングして投与する方法や、針に薬剤あるいは生体成分を透過させるための穴（中空針）や溝を形成する方法、針自身に薬剤を混合する方法等が提案されている。これらのマイクロニードルデバイスは、何れも高さ数十～数百マイクロメートル程度の非常に小さな突起物（マイクロニードル）を備えたものであるため、薬剤の適用方法によっては薬剤の経皮吸収性や吸収効率も大きく異なると考えられる。 In addition, various methods have been devised for applying drugs when using a device equipped with microneedles, such as a method of coating a drug on the surface of the microneedle and administering the drug or a biological component through the needle. A method of forming a hole (hollow needle) or a groove for making the needle, a method of mixing a drug in the needle itself, and the like have been proposed. Each of these microneedle devices has very small protrusions (microneedles) with a height of several tens to several hundreds of micrometers, so depending on the method of applying the drug, Absorption efficiency is also considered to differ greatly.
　例えば、マイクロニードルを用いて抗原（ワクチン）の経皮吸収性を効率的に促進させる方法として、マイクロニードル表面の一部分に薬剤をコーティングする方法があり、例えば非特許文献１に開示されている。これは、マイクロニードルの一部分（特に針部分のみ）に抗原（ワクチン）をコーティングした場合、適用した抗原（ワクチン）の全てまたはそのほとんどが体内へ移行し、正確な真皮への投与手段として有用であることを示している。 For example, as a method for efficiently promoting transdermal absorbability of an antigen (vaccine) using a microneedle, there is a method of coating a drug on a part of the surface of the microneedle, which is disclosed in Non-Patent Document 1, for example. This is useful when a part of the microneedle (especially only the needle part) is coated with an antigen (vaccine), or all or most of the applied antigen (vaccine) is transferred into the body and is useful as an accurate dermis administration means. It shows that there is.
　ところで、診断薬や薬剤のような医薬物質を効率的かつ安全に投与することの重要性が最近認識されてきている。特に最近は日本脳炎ウイルス抗原において急性散在性脳脊髄炎（ＡＤＥＭ）の問題が指摘されている通り（非特許文献２）であり、そのリスクを低減するために、サルの腎細胞由来のベロ細胞を用いた日本脳炎ウイルス抗原の開発が行われていて、いかに少ないウイルス抗原でいかに多くの人に免疫を惹起させるかという、ウイルス抗原の効率的で簡便な投与方法の開発が望まれる。 Incidentally, the importance of efficiently and safely administering pharmaceutical substances such as diagnostic agents and drugs has recently been recognized. In particular, as the problem of acute disseminated encephalomyelitis (ADEM) has recently been pointed out in Japanese encephalitis virus antigens (Non-patent Document 2), in order to reduce the risk, Vero cells derived from monkey kidney cells Japanese encephalitis virus antigens have been developed using, and it is desired to develop an efficient and simple administration method of virus antigens, such as how many humans can immunize with less virus antigens.
　特許文献１には、治療用物質の量を低減させ治療効果を達成する目的で中空針を持ったマイクロニードルによる投与が開示されているが、免疫の惹起についての記載はない。 Patent Document 1 discloses administration with a microneedle having a hollow needle for the purpose of reducing the amount of a therapeutic substance and achieving a therapeutic effect, but does not describe immunity induction.
特表２００６－５０６１０３号公報Special Table 2006-506103
　上述のように日本脳炎ウイルス抗原をマイクロニードルにコーティングして真皮への正確な投与を行うことにより、効率的で簡便な日本脳炎ウイルス抗原の投与の可能性があるにもかかわらず、これまで日本脳炎ウイルス抗原のマイクロニードルによる投与についての具体的な検討がなされていない。
　本発明の目的は、日本脳炎ウイルス抗原の免疫原性を増強させるマイクロニードルデバイスによる免疫原性増強方法を提供することにある。 Despite the possibility of efficient and simple administration of Japanese encephalitis virus antigen by coating Japanese encephalitis virus antigen on microneedles and performing accurate administration to the dermis as described above, No specific study has been made on the administration of encephalitis virus antigens using microneedles.
An object of the present invention is to provide a method for enhancing immunogenicity using a microneedle device that enhances the immunogenicity of a Japanese encephalitis virus antigen.
　本発明者は以上を技術背景として日々検討を重ねた結果、サル腎細胞（ベロ細胞）由来の抗原からなる日本脳炎ウイルス抗原をコーティングしたマイクロニードルを用いて日本脳炎ウイルス抗原を経皮投与することで、抗体性が上昇することを見いだした。更に日本脳炎ウイルス抗原をコーティングしたマイクロニードルを経皮投与後、ラウリルアルコールを含有する貼付剤を貼付することで、更なる効率的な抗体価の上昇が確認できた。 The present inventor has conducted daily studies with the above as a technical background, and as a result, transdermally administers Japanese encephalitis virus antigens using microneedles coated with Japanese encephalitis virus antigens composed of antigens derived from monkey kidney cells (Vero cells). And found that the antibody property increased. Furthermore, after the transdermal administration of microneedles coated with Japanese encephalitis virus antigen, a patch containing lauryl alcohol was affixed, whereby a further efficient increase in antibody titer could be confirmed.
　すなわち、本発明に係るマイクロニードルデバイスによる免疫原性増強方法は、サル腎細胞（ベロ細胞）由来の抗原からなる日本脳炎ウイルス抗原をコーティングしたポリ乳酸からなるマイクロニードルを備えたマイクロニードルデバイスを皮膚に当接し前記日本脳炎ウイルス抗原を経皮投与するものである。ここで、前記日本脳炎ウイルス抗原の経皮投与後、アジュバント効果を有するラウリルアルコールを含む貼付剤の貼付によりさらに免疫原性が増強される。その他にも皮膚浸透が可能なアジュバント活性を有する物質の塗布は免疫原性の増強効果が期待できる。 That is, the method for enhancing immunogenicity using a microneedle device according to the present invention is a method for treating a microneedle device comprising a microneedle made of polylactic acid coated with a Japanese encephalitis virus antigen made of an antigen derived from monkey kidney cells (Vero cells). And the Japanese encephalitis virus antigen is transdermally administered. Here, after the transdermal administration of the Japanese encephalitis virus antigen, the immunogenicity is further enhanced by applying a patch containing lauryl alcohol having an adjuvant effect. In addition, the application of a substance having an adjuvant activity that can penetrate the skin can be expected to enhance the immunogenicity.
　本発明に係るマイクロニードルデバイスは、サル腎細胞（ベロ細胞）由来の抗原からなる日本脳炎ウイルス抗原をコーティングしたポリ乳酸からなるマイクロニードルを備える。ここで、前記コーティングは、コーティング担体としてプルランを含むことが好ましい。 The microneedle device according to the present invention includes a microneedle composed of polylactic acid coated with a Japanese encephalitis virus antigen composed of an antigen derived from monkey kidney cells (Vero cells). Here, the coating preferably includes pullulan as a coating carrier.
　本発明によれば、サル腎細胞（ベロ細胞）由来の抗原からなる日本脳炎ウイルス抗原を、注射より簡便な操作で免疫原性を上昇させることができる。経皮的免疫賦活用のマイクロニードルデバイスを用いることにより、日本脳炎ウイルス抗原により誘発される免疫応答を刺激し、ウイルス抗原内の抗原の有効用量を減らすことができる。 According to the present invention, the immunogenicity can be increased by a simpler operation than injection of Japanese encephalitis virus antigen consisting of monkey kidney cell (Vero cell) -derived antigen. By using a microneedle device for transcutaneous immunostimulation, the immune response induced by Japanese encephalitis virus antigens can be stimulated, and the effective dose of antigens in the virus antigens can be reduced.
本発明に係るマイクロニードルデバイスの一例を示す図であり、（ａ）は斜視図、（ｂ）は（ａ）のＡ－Ｂ断面図である。It is a figure which shows an example of the microneedle device which concerns on this invention, (a) is a perspective view, (b) is AB sectional drawing of (a). 日本脳炎ウイルス抗原特異的ＩｇＧ抗体価の測定結果の一例を示すグラフである。It is a graph which shows an example of the measurement result of Japanese encephalitis virus antigen specific IgG antibody titer.
　図１は本発明に係るマイクロニードルデバイスの一例を示す図であり、（ａ）は斜視図、（ｂ）は（ａ）のＡ－Ｂ断面図である。図１（ａ）に示すように、本発明に係るマイクロニードルデバイス（インターフェイス）５は、マイクロニードル基板８と、皮膚又は粘膜を穿孔可能な二次元状に配置された複数のマイクロニードル６とを有する。マイクロニードル基板８は、各マイクロニードル６に対応して配置された複数の開口部７を備える。本例では、マイクロニードル６の形状は円錐状であるが、本発明はこれに限定されず、四角錐等の多角錐でもよく、また別の形状でもよい。また、複数のマイクロニードル６と複数の開口部７とが交互にそれぞれ正方格子状に配置されているが、本発明はこれに限定されない。さらに、マイクロニードル６と開口部７の数は図では１：１であるが、本発明はこれに限定されることなく、開口部７を含まないものも含む。 FIG. 1 is a view showing an example of a microneedle device according to the present invention, where (a) is a perspective view and (b) is a cross-sectional view taken along line AB of (a). As shown in FIG. 1 (a), a microneedle device (interface) 5 according to the present invention comprises a microneedle substrate 8 and a plurality of microneedles 6 arranged in a two-dimensional shape capable of perforating the skin or mucous membrane. Have. The microneedle substrate 8 includes a plurality of openings 7 arranged corresponding to the microneedles 6. In this example, the shape of the microneedle 6 is a conical shape, but the present invention is not limited to this, and may be a polygonal pyramid such as a quadrangular pyramid or another shape. Further, although the plurality of microneedles 6 and the plurality of openings 7 are alternately arranged in a square lattice shape, the present invention is not limited to this. Furthermore, although the number of the microneedles 6 and the openings 7 is 1: 1 in the drawing, the present invention is not limited to this, and includes those that do not include the openings 7.
　本例では、マイクロニードル６の一部又は全面が、サル腎細胞（ベロ細胞）由来の抗原からなる日本脳炎ウイルス抗原でコーティングされている。コーティング１は、例えば、図１（ｂ）に示すように、各マイクロニードル６の表面に配置される。コーティング１は、マイクロニードル６の全体に配置されているが、その一部に配置することもできる。用時に、図１（ａ）に示すマイクロニードル６の配置されたマイクロニードル基板面を皮膚に当接し、その反対面から薬物の溶解液を流すと、各開口部７から液体が流れ出て各マイクロニードル６に伝達され、上記日本脳炎ウイルス抗原が経皮吸収される。ここで、開口部７は必須ではなく、液体は開口部７を用いることなく別の手段でマイクロニードル６に供給してもよい。また、コーティング１は外部から液体を付与することなく、マイクロニードルを皮膚に穿孔したときの体液により溶解させることで、日本脳炎ウイルス抗原を皮膚内に放出させることができる。 In this example, a part or the whole of the microneedle 6 is coated with a Japanese encephalitis virus antigen composed of an antigen derived from monkey kidney cells (Vero cells). The coating 1 is arrange | positioned on the surface of each microneedle 6, for example, as shown in FIG.1 (b). The coating 1 is disposed on the entire microneedle 6, but may be disposed on a part thereof. At the time of use, when the microneedle substrate surface on which the microneedles 6 shown in FIG. 1 (a) are disposed is brought into contact with the skin and a drug solution is allowed to flow from the opposite surface, the liquid flows out from each opening 7 and each micro It is transmitted to the needle 6 and the Japanese encephalitis virus antigen is percutaneously absorbed. Here, the opening 7 is not essential, and the liquid may be supplied to the microneedle 6 by another means without using the opening 7. In addition, the coating 1 can release the Japanese encephalitis virus antigen into the skin by dissolving with a body fluid when the microneedle is perforated in the skin without applying a liquid from the outside.
　マイクロニードルデバイス中のマイクロニードルは、皮膚又は粘膜に穿刺されるマイクロニードル（針）とこの針部を支持する基板からなり、マイクロニードルは基板に複数配列されている。マイクロニードルは微小構造であり、マイクロニードルの高さ（長さ）ｈは、好ましくは５０μｍ～７００μｍであり、より好ましくは１００μｍ～６００μｍ、更に好ましくは２００μｍ～５００μｍである。ここで、マイクロニードルの長さを５０μｍ以上とするのは日本脳炎ウイルス抗原の経皮からの投与を確実とするためであり、７００μｍ以下とするのはマイクロニードルの神経との接触を回避し、痛みの可能性を確実に減少させることができると同時に出血の可能性を確実に回避するためである。また、その長さが７００μｍ以下であると、皮内に入る日本脳炎ウイルス抗原の量を効率よく投与することができる。 The microneedle in the microneedle device includes a microneedle (needle) that is punctured into the skin or mucous membrane and a substrate that supports the needle portion, and a plurality of microneedles are arranged on the substrate. The microneedle has a microstructure, and the height (length) h of the microneedle is preferably 50 μm to 700 μm, more preferably 100 μm to 600 μm, and further preferably 200 μm to 500 μm. Here, the length of the microneedle is set to 50 μm or more to ensure administration of the Japanese encephalitis virus antigen from the skin, and the length of 700 μm or less avoids contact of the microneedle with the nerve, This is because the possibility of pain can be surely reduced and at the same time the possibility of bleeding can be surely avoided. If the length is 700 μm or less, the amount of Japanese encephalitis virus antigen that enters the skin can be efficiently administered.
　ここで、マイクロニードルとは、凸状構造物であって広い意味での針形状又は針形状を含む構造物を意味し、円錐状構造の場合、通常その基底における直径は５０～２００μｍ程度である。また、マイクロニードルは、先鋭な先端を有する針形状のものに限定されるものではなく、先の尖っていない形状も含むものである。マイクロニードルは、好適には非金属製の合成または天然の樹脂素材を用いて作製される。また、マイクロニードルの形状は本例では円錐状であるが、本発明はこれに限定されず、四角錐等の多角錐でもよく、また別の形状でもよい。 Here, the microneedle means a convex structure and a needle shape in a broad sense or a structure including a needle shape. In the case of a conical structure, the diameter at the base is usually about 50 to 200 μm. . Further, the microneedle is not limited to a needle shape having a sharp tip, and includes a shape having no sharp tip. The microneedles are preferably made using a non-metallic synthetic or natural resin material. Further, the shape of the microneedle is a conical shape in this example, but the present invention is not limited to this, and may be a polygonal pyramid such as a quadrangular pyramid or another shape.
マイクロニードル基板はマイクロニードルを支持するための土台であり、その形態は限定されるものではなく、例えば図１のように貫通した穴（開口部）を備えた基板であってもよく、この場合、基板の背面から、マイクロニードルにコーティングした日本脳炎ウイルス抗原の溶解液を流すことができる他、日本脳炎ウイルス抗原を開口部およびマイクロニードルを介して流して投与することもできる。マイクロニードルあるいは基板の材質としては、シリコン、二酸化ケイ素、セラミック、金属（ステンレス、チタン、ニッケル、モリブテン、クロム、コバルト等）及び合成または天然の樹脂素材等が挙げられるが、マイクロニードルの抗原性および材質の単価を考慮すると、ポリ乳酸、ポリグリコリド、ポリ乳酸－ｃｏ－ポリグリコリド、プルラン、カプロノラクトン、ポリウレタン、ポリ無水物等の生分解性ポリマーや、非分解性ポリマーであるポリカーボネート、ポリメタクリル酸、エチレンビニルアセテート、ポリテトラフルオロエチレン、ポリオキシメチレン等の合成または天然の樹脂素材が特に好ましい。また、多糖類であるヒアルロン酸、プルラン、デキストラン、デキストリン若しくはコンドロイチン硫酸等も好適である。特に、ポリ乳酸は生分解性樹脂であり、インプラント製剤としても使用実績がある（特表２００２－５１７３００号公報または、Ｊｏｕｒｎａｌ　ｏｆ　Ｃｏｎｔｒｏｌｌｅｄ　Ｒｅｌｅａｓｅ　１０４（２００５）５１－６６）ことから、強度面、安全性からみて最も好適なマイクロニードル素材の一つである。 The microneedle substrate is a base for supporting the microneedle, and the form thereof is not limited. For example, the substrate may be a substrate having a through hole (opening) as shown in FIG. In addition to allowing the solution of Japanese encephalitis virus antigen coated on the microneedle to flow from the back of the substrate, the Japanese encephalitis virus antigen can also be administered by flowing through the opening and the microneedle. Examples of the material of the microneedle or the substrate include silicon, silicon dioxide, ceramic, metal (stainless steel, titanium, nickel, molybdenum, chromium, cobalt, etc.) and synthetic or natural resin materials. Considering the unit price of the material, biodegradable polymers such as polylactic acid, polyglycolide, polylactic acid-co-polyglycolide, pullulan, capronolactone, polyurethane, polyanhydride, polycarbonates that are non-degradable polymers, polymethacrylic acid, A synthetic or natural resin material such as ethylene vinyl acetate, polytetrafluoroethylene, or polyoxymethylene is particularly preferable. Also suitable are polysaccharides such as hyaluronic acid, pullulan, dextran, dextrin or chondroitin sulfate. In particular, polylactic acid is a biodegradable resin and has been used as an implant preparation (Japanese Patent Publication No. 2002-517300 or Journal of Controlled Release 104 (2005) 51-66). It is one of the most suitable microneedle materials in view of the above.
　マイクロニードル（針）の密度は、典型的には、針の横列は１ミリメートル（ｍｍ）当たり約１ないし１０の密度が提供される様に横列間が空けられている。一般に、横列は横列内の針の空間に対し実質等しい距離だけ離れており、１ｃｍ２当たり１００～１００００本の針密度を有し、好ましくは１００～５０００本、より好ましくは２００～２０００本、更に好ましくは４００～１０００本である。１００本以上の針密度があると、効率良く皮膚を穿孔することができ、１００００本を超える針密度では、マイクロニードルに皮膚穿孔可能な強度を付与することが難しくなる。 The density of microneedles (needles) is typically spaced between the rows such that the rows of needles provide a density of about 1 to 10 per millimeter (mm). In general, the rows are separated by a substantially equal distance from the needle space in the row and have a needle density of 100-10000 per cm 2 , preferably 100-5000, more preferably 200-2000, and even more The number is preferably 400 to 1000. When there is a needle density of 100 or more, the skin can be efficiently perforated, and when the needle density exceeds 10,000, it is difficult to give the microneedles strength capable of perforating the skin.
　マイクロニードルの製法としては、シリコン基板を用いたウエットエッチング加工又はドライエッチング加工、金属又は樹脂を用いた精密機械加工（放電加工、レーザー加工、ダイシング加工、ホットエンボス加工、射出成型加工等）、機械切削加工等が挙げられる。これらの加工法により、針部と支持部は、一体に成型される。針部を中空にする方法としては、針部を作製後、レーザー加工等で２次加工する方法が挙げられる。 Microneedle manufacturing methods include wet etching or dry etching using a silicon substrate, precision machining using metal or resin (electric discharge machining, laser machining, dicing, hot embossing, injection molding, etc.), machinery Examples include cutting. By these processing methods, the needle portion and the support portion are integrally molded. As a method for making the needle part hollow, there is a method of performing secondary processing by laser processing or the like after producing the needle part.
　マイクロニードルへコーティングを行う際に、コーティング液の溶媒揮発による薬剤の濃度変化および物性の変化を最小限にするために、装置の設置環境の温湿度を一定に制御することもできる。溶媒の蒸散を防ぐためには、温度を下げるか湿度を上げるかのどちらかまたはその両方を制御することが好ましい。温度を制御しない場合の室温での湿度は、相対湿度として５０～１００％ＲＨであり、好ましくは７０．０～１００％ＲＨである。５０％ＲＨ以下であると溶媒の蒸発が起こり、コーティング液の物性の変化が起こる場合がある。加湿方式には、目的の湿度状態が確保できれば特に限定されないが、気化式、蒸気式、水噴霧式などがある。 When coating the microneedles, the temperature and humidity of the installation environment of the apparatus can be controlled to be constant in order to minimize changes in drug concentration and physical properties due to solvent volatilization of the coating solution. In order to prevent transpiration of the solvent, it is preferable to control either the temperature or the humidity or both. When the temperature is not controlled, the humidity at room temperature is 50 to 100% RH as relative humidity, and preferably 70.0 to 100% RH. If it is 50% RH or less, the solvent may evaporate and the physical properties of the coating solution may change. The humidification method is not particularly limited as long as the target humidity state can be secured, but there are a vaporization method, a steam method, a water spray method, and the like.
　本発明で用いる日本脳炎ウイルス抗原の調製は、例えばＷＯ０１／０７６６２４号を参考に行うことができる。 Preparation of the Japanese encephalitis virus antigen used in the present invention can be performed with reference to, for example, WO01 / 076664.
　マイクロニードルにコーティングするコーティング液は、日本脳炎ウイルス抗原の他、コーティング担体及び液体組成物を含み得る。また本発明コーティングとは、マイクロニードル（針）にコーティング液が留まり固着した状態が好ましく、そのためにコーティング液に乾燥工程を加えて固着させることもある。 The coating liquid for coating the microneedles can contain a coating carrier and a liquid composition in addition to the Japanese encephalitis virus antigen. The coating of the present invention is preferably a state in which the coating liquid stays and adheres to the microneedles (needles). For this purpose, the coating liquid may be fixed by applying a drying step.
　コーティング担体としては、日本脳炎ウイルス抗原と比較的相溶性（均一に交わる性質）のある多糖類の担体が好ましく、ポリヒドロキシメチルセルロース、ヒドロキシプロピルセルロース、ポリヒドロキシプロピルメチルセルロース、ポリメチルセルロース、デキストラン、ポリエチレングリコール、プルラン、カルメロースナトリウム、コンドロイチン硫酸、ヒアルロン酸、デキストラン、アラビアゴム等が好ましく、更にヒドロキシプロピルセルロース、プルラン、アラビアゴムがより好ましい。また、ヒドロキシプロピルセルロース（ＨＰＣ－ＳＳＬ（分子量：１５，０００～３０，０００）、ＨＰＣ－ＳＬ（分子量：３０，０００～５０，０００）、ＨＰＣ－Ｌ（分子量：５５，０００～７０，０００）、ＨＰＣ－Ｍ（分子量：１１０，０００～１５０，０００）、ＨＰＣ－Ｈ（分子量：２５０，０００～４００，０００））、プルラン、ヒアルロン酸が更に好ましい。特に、日本脳炎ウイルス抗原との相溶性の面でプルランが最も好ましい。 As the coating carrier, a polysaccharide carrier that is relatively compatible with the Japanese encephalitis virus antigen (a property that uniformly crosses) is preferable. Polyhydroxymethylcellulose, hydroxypropylcellulose, polyhydroxypropylmethylcellulose, polymethylcellulose, dextran, polyethylene glycol, Pullulan, carmellose sodium, chondroitin sulfate, hyaluronic acid, dextran, gum arabic and the like are preferred, and hydroxypropyl cellulose, pullulan and gum arabic are more preferred. Further, hydroxypropylcellulose (HPC-SSL (molecular weight: 15,000 to 30,000), HPC-SL (molecular weight: 30,000 to 50,000), HPC-L (molecular weight: 55,000 to 70,000) HPC-M (molecular weight: 110,000 to 150,000), HPC-H (molecular weight: 250,000 to 400,000)), pullulan and hyaluronic acid are more preferred. In particular, pullulan is most preferable in terms of compatibility with the Japanese encephalitis virus antigen.
　コーティング液全体のコーティング担体の含量は、１～７０重量％であり、好ましくは１～４０重量％であり、特に好ましくは３～２５重量％である。また、このコーティング担体は、液だれすることのないようある程度の粘性が必要である場合があり、粘度として１００～１０００００ｃｐｓ程度必要である。より好ましい粘度は、５００～６００００ｃｐｓである。粘度がこの範囲にあることにより、マイクロニードルの材質に依存することなく、所望量のコーティング液を一度に塗布することが可能となる。また、一般的に粘度が高くなればなるほどコーティング液の量が増える傾向になる。 The content of the coating carrier in the entire coating liquid is 1 to 70% by weight, preferably 1 to 40% by weight, particularly preferably 3 to 25% by weight. In addition, this coating carrier may require a certain degree of viscosity so that it does not drip, and the viscosity is required to be about 100 to 100,000 cps. A more preferred viscosity is 500 to 60000 cps. When the viscosity is in this range, it is possible to apply a desired amount of the coating liquid at a time without depending on the material of the microneedle. In general, the higher the viscosity, the greater the amount of coating solution.
　マイクロニードルをコーティングするのに使用される液体組成物は、生体適合性の担体、送達されるべき日本脳炎ウイルス抗原、および場合によってはいずれかのコーティング補助物質を揮発性液体と混合することにより調製する。揮発性液体は、水、ジメチルスルホキシド、ジメチルホルムアミド、エタノール、イソプロピルアルコールおよびそれらの混合物等であることができる。これらの中で水が最も好ましい。液体のコーティング液もしくは懸濁液は、典型的には、０．１～６５重量％の日本脳炎ウイルス抗原濃度を有することができ、好ましくは１～３０重量％、更に好ましくは、３～２０重量％である。
　他の既知の製剤補助物質は、それらがコーティングの必要な溶解性および粘度の特徴ならびに乾燥されたコーティングの性状および物性に有害な影響を及ぼさない限りは、コーティングに添加してもよい。 The liquid composition used to coat the microneedles is prepared by mixing a biocompatible carrier, the Japanese encephalitis virus antigen to be delivered, and possibly any coating aids with a volatile liquid. To do. The volatile liquid can be water, dimethyl sulfoxide, dimethylformamide, ethanol, isopropyl alcohol, mixtures thereof, and the like. Of these, water is most preferred. The liquid coating solution or suspension can typically have a Japanese encephalitis virus antigen concentration of 0.1 to 65% by weight, preferably 1 to 30% by weight, more preferably 3 to 20% by weight. %.
Other known formulation adjuvants may be added to the coating as long as they do not adversely affect the required solubility and viscosity characteristics of the coating and the properties and physical properties of the dried coating.
　マイクロニードルのコーティングの厚さは、５０μｍ未満であり、好ましくは２５μｍ未満、さらに好ましくは１～１０μｍである。一般に、コーティングの厚さは、乾燥後にマイクロニードルの表面にわたって測定される平均の厚さである。コーティングの厚さは、一般に、コーティング担体の複数の被膜を適用することにより増大させること、すなわち、コーティング担体固着後にコーティング工程をくり返すことで増大させることができる。 The thickness of the microneedle coating is less than 50 μm, preferably less than 25 μm, more preferably 1 to 10 μm. In general, the coating thickness is the average thickness measured across the surface of the microneedle after drying. The thickness of the coating can generally be increased by applying multiple coatings of the coating carrier, i.e. by repeating the coating process after fixing the coating carrier.
　マイクロニードルの高さ（長さ）ｈは、上述のとおり、好ましくは５０μｍ～７００μｍである。マイクロニードルのコーティングの高さＨは、マイクロニードルの高さｈによって変動するが、０μｍ～７００μｍの範囲とすることができ、通常１０μｍ～７００μｍの範囲内であり、好ましくは、３０μｍ～５００μｍ程度である。 The height (length) h of the microneedle is preferably 50 μm to 700 μm as described above. The coating height H of the microneedle varies depending on the height h of the microneedle, but can be in the range of 0 μm to 700 μm, usually in the range of 10 μm to 700 μm, and preferably about 30 μm to 500 μm. is there.
　本発明のマイクロニードルデバイスを使用して日本脳炎ウイルス抗原による免疫の惹起を増強するために、例えば、アジュバント効果のある脂肪族アルコール類を本発明マイクロニードルデバイスにより投与した部位に塗布することができる。このような脂肪族アルコール類においては、直鎖または分枝状の脂肪族アルコール類が好ましい。このような脂肪族アルコール類において、炭素数および分子量に特に限定はないが、皮膚透過性を考慮すると、炭素数８～２０であることがより好ましい。また、かかる脂肪族アルコール類は、飽和または不飽和のいずれであってもよい。
　これら脂肪族アルコール類の中には経皮吸収における吸収促進剤として利用されることも多いが、本発明における脂肪族アルコール類は、ＷＯ２００７／０１５４４１を参照すると吸収促進作用に加えて、アジュバント効果を期待することができる。 In order to enhance the induction of immunity by Japanese encephalitis virus antigen using the microneedle device of the present invention, for example, an aliphatic alcohol having an adjuvant effect can be applied to the site administered by the microneedle device of the present invention. . Of these aliphatic alcohols, linear or branched aliphatic alcohols are preferred. In such aliphatic alcohols, the carbon number and molecular weight are not particularly limited, but considering the skin permeability, it is more preferably 8 to 20 carbon atoms. Such aliphatic alcohols may be saturated or unsaturated.
Among these fatty alcohols, they are often used as absorption enhancers in percutaneous absorption. However, referring to WO2007 / 015441, the aliphatic alcohols in the present invention have an adjuvant effect in addition to the absorption promoting action. You can expect.
　そのような脂肪族アルコール類は、例えばオクチルドデカノール、ラウリルアルコール、オレイルアルコール、イソステアリルアルコール、デカノールなどであるが、その中でもラウリルアルコール、オレイルアルコール、イソステアリルアルコールが特に好ましく、ラウリルアルコールが最も好ましい。 Such aliphatic alcohols are, for example, octyldodecanol, lauryl alcohol, oleyl alcohol, isostearyl alcohol, decanol, etc. Among them, lauryl alcohol, oleyl alcohol, and isostearyl alcohol are particularly preferable, and lauryl alcohol is most preferable. .
　ウイルス抗原に混ぜ合わせる場合には、本発明の脂肪族アルコール類は０．１～９９重量％で好ましく配合され、５～９０重量％で配合されるのがより好ましく、とくに１０～８０重量％であるとさらに好ましい。最も好ましい組成中における脂肪族アルコール類の含量は１５～７５重量％である。 When mixed with a viral antigen, the aliphatic alcohol of the present invention is preferably blended at 0.1 to 99% by weight, more preferably 5 to 90% by weight, particularly 10 to 80% by weight. More preferably. The content of aliphatic alcohols in the most preferred composition is 15 to 75% by weight.
　該アジュバントを含む医薬製剤の形態としては、経皮的に投与できる形態であれば特に限定されないが、パップ剤やパッチ製剤等の貼付剤、軟膏剤、クリーム剤、液剤、ゲル剤、ローション剤などから必要に応じて選択できるが、特にパッチ製剤が好ましい。
　パッチ製剤の投与部位については特に限定されないが、抗原の投与部位に近い方が好ましく、抗原の投与部位の上部に貼付することが更に好ましい。
　該経皮投与製剤は、基剤として溶解剤、溶解補助剤、ｐＨ調整剤、防腐剤、吸収促進剤、安定化剤、充填剤、増粘剤、粘着剤、湿潤剤などの任意の成分を、該アジュバントとを組み合わせて用いることによって、常法により製造することができる。 The form of the pharmaceutical preparation containing the adjuvant is not particularly limited as long as it can be administered transdermally. However, patches, ointments, creams, solutions, gels, lotions, etc. However, a patch preparation is particularly preferable.
The administration site of the patch preparation is not particularly limited, but is preferably closer to the antigen administration site, and more preferably applied to the upper part of the antigen administration site.
The preparation for transdermal administration contains, as a base, optional components such as a solubilizer, a solubilizer, a pH adjuster, an antiseptic, an absorption accelerator, a stabilizer, a filler, a thickener, an adhesive, and a wetting agent. By using the adjuvant in combination, it can be produced by a conventional method.
　該アジュバントを含む医薬製剤の組成は、０．１～９９重量％で好ましく配合され、５～９０重量％で配合されるのがより好ましく、とくに１０～８０重量％であるとさらに好ましい。最も好ましい組成中における脂肪族アルコール類の含量は１５～７５重量％である。 The composition of the pharmaceutical preparation containing the adjuvant is preferably 0.1 to 99% by weight, more preferably 5 to 90% by weight, and even more preferably 10 to 80% by weight. The content of aliphatic alcohols in the most preferred composition is 15 to 75% by weight.
（実験例１）
　以下の通り調整したサル腎細胞（ベロ細胞）由来の抗原を含む日本脳炎ウイルス抗原をＢＩＯＭＡＸ－１０Ｋ（ミリポア社製）を用い、遠心分離により濃縮し、高分子ポリマー（プルラン）と混合の後、加湿器にて相対湿度９０～１００％ＨＲを維持しながら、マイクロニードルデバイスのポリ乳酸製マイクロニードル（高さ約３００μｍ、密度８４１本／ｃｍ２、四角錐形状）にコーティングした。コーティング含量は抗原２μｇ／ｐａｔｃｈとし、４週齢のｄｄＹマウス（メス）に麻酔下で腹部剃毛後、コーティングマイクロニードルを皮膚に２時間穿刺投与した（４例）。アジュバント投与群（５例）は、アジュバント（ラウリルアルコール）貼付剤をマイクロニードル投与部位上部に貼付した。１週間後に同条件でブーストし、その１週間後に採血後、日本脳炎ウイルス抗原特異的ＩｇＧ抗体価を測定した。図２にその結果を示す。 (Experimental example 1)
Japanese encephalitis virus antigens containing antigens derived from monkey kidney cells (Vero cells) prepared as follows were concentrated by centrifugation using BIOMAX-10K (Millipore), mixed with a polymer (pullulan), While maintaining a relative humidity of 90 to 100% HR with a humidifier, the microneedle made of polylactic acid (height of about 300 μm, density of 841 pieces / cm 2 , square pyramid shape) was coated. The coating content was 2 μg / patch of antigen, and a 4-week-old ddY mouse (female) was shaved from the abdomen under anesthesia, and then the coated microneedle was punctured for 2 hours into the skin (4 cases). In the adjuvant administration group (5 cases), an adjuvant (lauryl alcohol) patch was affixed to the upper part of the microneedle administration site. One week later, boosting was performed under the same conditions. After one week of blood collection, the Japanese encephalitis virus antigen-specific IgG antibody titer was measured. The results are shown in FIG.
（抗体価の測定）
　採血した血液を４℃にて一夜静置した後、遠心により血清を分離し、非働化（５６℃、３０分間処理）した。コーティングバッファーで５μｇ／ｍＬに希釈したウイルス抗原を１００μＬ／ｗｅｌｌにてｗｅｌｌに添加し、４℃、一夜静置した。固相化したプレートを洗浄バッファー４００μＬ／ｗｅｌｌで３回洗浄し、ブロッキングバッファーを２５０μＬ／ｗｅｌｌ添加し、３７℃、１時間反応した。その後、洗浄バッファー４００μＬ／ｗｅｌｌで３回洗浄し、十分に液を切った後に希釈バッファーにて１００倍希釈し、２倍系列で段階希釈した検体を各１００μＬ／ｗｅｌｌ添加し、３７℃、２時間反応させた。
　次に、洗浄バッファー４００μＬ／ｗｅｌｌで３回洗浄し、希釈したＨＲＰ標識抗体１００μＬ／ｗｅｌｌを添加し、３７℃、９０分間反応させた。バッファー４００μＬ／ｗｅｌｌで３回洗浄し、ＴＭＢＺ溶液を１００μＬ／ｗｅｌｌ添加し、暗所にて室温、３０分間反応後、０．３Ｎ　硫酸を１００μＬ／ｗｅｌｌ添加して反応を停止し、４５０ｎｍの吸光度を測定した。
　コーティングバッファー；０．０５Ｍ炭酸バッファー（ｐＨ９．５）
　洗浄バッファー；０．０５％Ｔｗｅｅｎ２０含有ＰＢＳ（ＰＢＳ－Ｔ）
　ブロッキングバッファー；０．５％ＢＳＡ含有ＰＢＳ
　希釈バッファー；０．５％ＢＳＡ含有ＰＢＳ－Ｔ (Measurement of antibody titer)
The collected blood was allowed to stand overnight at 4 ° C., and then the serum was separated by centrifugation and inactivated (treated at 56 ° C. for 30 minutes). The virus antigen diluted to 5 μg / mL with the coating buffer was added to the well at 100 μL / well and allowed to stand at 4 ° C. overnight. The solid-phased plate was washed 3 times with 400 μL / well of washing buffer, 250 μL / well of blocking buffer was added, and reacted at 37 ° C. for 1 hour. Thereafter, the sample was washed 3 times with a washing buffer of 400 μL / well, sufficiently drained, then diluted 100-fold with a dilution buffer, and 100 μL / well of each sample diluted serially in a 2-fold series was added at 37 ° C. for 2 hours. Reacted.
Next, the plate was washed 3 times with a washing buffer of 400 μL / well, diluted HRP-labeled antibody 100 μL / well was added, and reacted at 37 ° C. for 90 minutes. Wash 3 times with 400 μL / well of buffer, add 100 μL / well of TMBZ solution, react at room temperature for 30 minutes in the dark, then add 100 μL / well of 0.3 N sulfuric acid to stop the reaction, and absorb the absorbance at 450 nm. It was measured.
Coating buffer; 0.05M carbonate buffer (pH 9.5)
Washing buffer; PBS containing 0.05% Tween 20 (PBS-T)
Blocking buffer; PBS containing 0.5% BSA
Dilution buffer: PBS-T containing 0.5% BSA
　図２に示すように、マイクロニードルデバイス（ＭＮ）単独において十分な日本脳炎ウイルス抗原特異的ＩｇＧ抗体価が得られ、更にアジュバント（ラウリルアルコール）貼付剤を併用した場合（ＭＮ＋ＬＡｔａｐｅ）には更に高い日本脳炎ウイルス抗原特異的ＩｇＧ抗体価を得ることができる。 As shown in FIG. 2, a sufficient Japanese encephalitis virus antigen-specific IgG antibody titer can be obtained with the microneedle device (MN) alone, and even higher when the adjuvant (lauryl alcohol) patch is used together (MN + LAtape). Encephalitis virus antigen-specific IgG antibody titers can be obtained.
　本発明は、以下のものを含む。
（１）基板に二次元状に配置され、皮膚を穿孔可能なポリ乳酸からなる、複数のマイクロニードルを備え、該マイクロニードルに、サル腎細胞（ベロ細胞）由来の日本脳炎ウイルス抗原がコーティングされたマイクロニードルデバイス。
（２）前記マイクロニードルが円錐型または角錐型である、上記（１）に記載のマイクロニードルデバイス。
（３）前記コーティングが、コーティング担体としてプルランを含む、上記（１）または（２）に記載のマイクロニードルデバイス。
（４）前記コーティングが、室温での相対湿度７０．０～１００％ＲＨにて行われる、上記（１）から（３）のいずれかに記載のマイクロニードルデバイス。
（５）前記コーティングが、アジュバント活性を有する物質を含む、上記（１）から（４）のいずれかに記載のマイクロニードルデバイス。
（６）前記アジュバント活性を有する物質が、ラウリルアルコールである、上記（５）に記載のマイクロニードルデバイス。
（７）前記マイクロニードルデバイスの基板が、日本脳炎ウイルス抗原液または日本脳炎ウイルス抗原溶解液を伝達可能な複数の開口部を有するものである、上記（１）から（６）のいずれかに記載のマイクロニードルデバイス。
（８）前記マイクロニードルの高さが２００～５００μｍである、上記（１）から（７）のいずれかに記載のマイクロニードルデバイス。
（９）前記マイクロニードルが４００～１０００本／ｃｍ２の密度で配置される、上記（１）から（８）のいずれかに記載のマイクロニードルデバイス。
（１０）マイクロニードルデバイスにより日本脳炎ウイルス抗原を投与後、更にアジュバント活性を有する物質を含有する貼付剤を貼付して、日本脳炎ウイルス抗原の奏効性を上昇させる方法において、当該方法に用いられるマイクロニードルデバイスであって、基板に二次元状に配置され、皮膚を穿孔可能なポリ乳酸からなる複数のマイクロニードルを有し、当該マイクロニードルは、サル腎細胞（ベロ細胞）由来の日本脳炎ウイルス抗原がコーティングされていることを特徴とするマイクロニードルデバイス。
（１１）基板に二次元状に配置され、皮膚を穿孔可能なポリ乳酸からなる、複数のマイクロニードルを備え、該マイクロニードルにサル腎細胞（ベロ細胞）由来の日本脳炎ウイルス抗原をコーティングしてなるマイクロニードルデバイスにより、日本脳炎ウイルス抗原の奏効性を上昇させる方法。
（１２）前記マイクロニードルが円錐型または角錐型である、上記（１１）に記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏功性を上昇させる方法。
（１３）前記コーティングが、コーティング担体としてプルランを含む、上記（１１）または（１２）に記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。
（１４）前記コーティングが、室温での相対湿度７０．０～１００％ＲＨにて行われる、請求項１１から１３のいずれかに記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。
（１５）前記コーティングが、アジュバント活性を有する物質を含む、上記（１１）から（１４）のいずれかに記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。
（１６）前記アジュバント活性を有する物質が、ラウリルアルコールである、上記（１５）に記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。
（１７）前記マイクロニードルデバイスの基板が、日本脳炎ウイルス抗原液または日本脳炎ウイルス抗原溶解液を伝達可能な複数の開口部を有するものである、上記（１１）から（１６）のいずれかに記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。
（１８）前記マイクロニードルの高さが２００～５００μｍである、上記（１１）から（１７）のいずれかに記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。
（１９）前記マイクロニードルが４００～１０００本／ｃｍ２の密度で配置される、上記（１１）から（１８）のいずれかに記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。
（２０）上記（１１）から（１９）のいずれかに記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法による日本脳炎ウイルス抗原の経皮投与後、更にアジュバント活性を有する貼付剤を貼付して、更なるマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。
（２１）前記貼付剤のアジュバント活性を有する物質が、ラウリルアルコールである、上記（２０）に記載の更なるマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。 The present invention includes the following.
(1) A plurality of microneedles that are two-dimensionally arranged on a substrate and are made of polylactic acid capable of perforating the skin, and coated with Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells). Microneedle device.
(2) The microneedle device according to (1) above, wherein the microneedle is conical or pyramidal.
(3) The microneedle device according to (1) or (2), wherein the coating contains pullulan as a coating carrier.
(4) The microneedle device according to any one of (1) to (3), wherein the coating is performed at a relative humidity of 70.0 to 100% RH at room temperature.
(5) The microneedle device according to any one of (1) to (4), wherein the coating contains a substance having an adjuvant activity.
(6) The microneedle device according to (5) above, wherein the substance having adjuvant activity is lauryl alcohol.
(7) The substrate of the microneedle device according to any one of (1) to (6), wherein the substrate has a plurality of openings through which a Japanese encephalitis virus antigen solution or a Japanese encephalitis virus antigen solution can be transmitted. Microneedle device.
(8) The microneedle device according to any one of (1) to (7), wherein the height of the microneedle is 200 to 500 μm.
(9) The microneedle device according to any one of (1) to (8), wherein the microneedles are arranged at a density of 400 to 1000 needles / cm 2 .
(10) A method for increasing the response of Japanese encephalitis virus antigen by administering a patch containing a substance having an adjuvant activity after administration of Japanese encephalitis virus antigen using a microneedle device. A needle device having a plurality of microneedles made of polylactic acid that is two-dimensionally arranged on a substrate and can perforate the skin, and the microneedles are Japanese encephalitis virus antigens derived from monkey kidney cells (Vero cells) Is coated with a microneedle device.
(11) A plurality of microneedles made of polylactic acid that is two-dimensionally arranged on a substrate and capable of perforating the skin, and the microneedles are coated with a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells). A method for increasing the response of Japanese encephalitis virus antigens using the microneedle device.
(12) The method for increasing the efficacy of a Japanese encephalitis virus antigen by the microneedle device according to (11) above, wherein the microneedle is a cone type or a pyramid type.
(13) The method for increasing the response of Japanese encephalitis virus antigen by the microneedle device according to (11) or (12), wherein the coating contains pullulan as a coating carrier.
(14) The method for increasing the response of Japanese encephalitis virus antigen by the microneedle device according to any one of claims 11 to 13, wherein the coating is performed at a relative humidity of 70.0 to 100% RH at room temperature. .
(15) The method for increasing the response of Japanese encephalitis virus antigen by the microneedle device according to any one of (11) to (14), wherein the coating contains a substance having an adjuvant activity.
(16) The method for increasing the efficacy of a Japanese encephalitis virus antigen by the microneedle device according to (15), wherein the substance having an adjuvant activity is lauryl alcohol.
(17) The substrate of the microneedle device according to any one of (11) to (16), wherein the substrate has a plurality of openings capable of transmitting Japanese encephalitis virus antigen solution or Japanese encephalitis virus antigen solution. To increase the response of Japanese encephalitis virus antigens using the microneedle device.
(18) The method for increasing the response of Japanese encephalitis virus antigen by the microneedle device according to any one of (11) to (17), wherein the microneedle has a height of 200 to 500 μm.
(19) The method for increasing the response of Japanese encephalitis virus antigen by the microneedle device according to any one of (11) to (18), wherein the microneedles are arranged at a density of 400 to 1000 / cm 2. .
(20) A patch having further adjuvant activity after transdermal administration of Japanese encephalitis virus antigen by the method for increasing the efficacy of Japanese encephalitis virus antigen by the microneedle device according to any of (11) to (19) above A method of increasing the response of Japanese encephalitis virus antigens with a further microneedle device.
(21) The method for increasing the efficacy of a Japanese encephalitis virus antigen by the further microneedle device according to (20), wherein the substance having the adjuvant activity of the patch is lauryl alcohol.
　本発明は、マイクロニードルデバイスを用いることにより、効率的で、しかも簡便な操作でサル腎細胞（ベロ細胞）由来の日本脳炎ウイルス抗原からなる日本脳炎ウイルス抗原の抗原性を上昇させることを可能とするものであり、産業上の利用可能性がある。 The present invention makes it possible to increase the antigenicity of a Japanese encephalitis virus antigen comprising a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells) by an efficient and simple operation by using a microneedle device. And has industrial applicability.

Claims (21)

  1. 　基板に二次元状に配置され、皮膚を穿孔可能なポリ乳酸からなる、複数のマイクロニードルを備え、該マイクロニードルに、サル腎細胞（ベロ細胞）由来の日本脳炎ウイルス抗原がコーティングされたマイクロニードルデバイス。 A microneedle comprising a plurality of microneedles that are two-dimensionally arranged on a substrate and made of polylactic acid capable of perforating the skin, and which is coated with a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells) device.
  2. 　前記マイクロニードルが円錐型または角錐型である、請求項１に記載のマイクロニードルデバイス。 The microneedle device according to claim 1, wherein the microneedle is conical or pyramidal.
  3. 　前記コーティングが、コーティング担体としてプルランを含む、請求項１または２に記載のマイクロニードルデバイス。 The microneedle device according to claim 1 or 2, wherein the coating contains pullulan as a coating carrier.
  4. 　前記コーティングが、室温での相対湿度７０．０～１００％ＲＨにて行われる、請求項１から３のいずれかに記載のマイクロニードルデバイス。 The microneedle device according to any one of claims 1 to 3, wherein the coating is performed at a relative humidity of 70.0 to 100% RH at room temperature.
  5. 　前記コーティングが、アジュバント活性を有する物質を含む、請求項１から４のいずれかに記載のマイクロニードルデバイス。 The microneedle device according to any one of claims 1 to 4, wherein the coating contains a substance having an adjuvant activity.
  6. 　前記アジュバント活性を有する物質が、ラウリルアルコールである、請求項５に記載のマイクロニードルデバイス。 The microneedle device according to claim 5, wherein the substance having adjuvant activity is lauryl alcohol.
  7. 　前記マイクロニードルデバイスの基板が、日本脳炎ウイルス抗原液または日本脳炎ウイルス抗原溶解液を伝達可能な複数の開口部を有するものである、請求項１から６のいずれかに記載のマイクロニードルデバイス。 The microneedle device according to any one of claims 1 to 6, wherein the substrate of the microneedle device has a plurality of openings capable of transmitting Japanese encephalitis virus antigen solution or Japanese encephalitis virus antigen solution.
  8. 　前記マイクロニードルの高さが２００～５００μｍである、請求項１から７のいずれかに記載のマイクロニードルデバイス。 The microneedle device according to any one of claims 1 to 7, wherein a height of the microneedle is 200 to 500 µm.
  9. 　前記マイクロニードルが４００～１０００本／ｃｍ２の密度で配置される、請求項１から８のいずれかに記載のマイクロニードルデバイス。 The microneedles are arranged in a density of 400 to 1000 / cm 2, the microneedle device according to any of claims 1 to 8.
  10. 　マイクロニードルデバイスにより日本脳炎ウイルス抗原を投与後、更にアジュバント活性を有する物質を含有する貼付剤を貼付して、日本脳炎ウイルス抗原の奏効性を上昇させる方法において、当該方法に用いられるマイクロニードルデバイスであって、基板に二次元状に配置され、皮膚を穿孔可能なポリ乳酸からなる複数のマイクロニードルを有し、当該マイクロニードルは、サル腎細胞（ベロ細胞）由来の日本脳炎ウイルス抗原がコーティングされていることを特徴とするマイクロニードルデバイス。 After administering Japanese encephalitis virus antigen using a microneedle device, a patch containing a substance having an adjuvant activity is further applied to increase the response of Japanese encephalitis virus antigen. A plurality of microneedles made of polylactic acid that are two-dimensionally arranged on a substrate and can perforate the skin, and the microneedles are coated with a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells). A microneedle device characterized by that.
  11. 　基板に二次元状に配置され、皮膚を穿孔可能なポリ乳酸からなる、複数のマイクロニードルを備え、該マイクロニードルにサル腎細胞（ベロ細胞）由来の日本脳炎ウイルス抗原をコーティングしてなるマイクロニードルデバイスにより、日本脳炎ウイルス抗原の奏効性を上昇させる方法。 A microneedle comprising a plurality of microneedles that are two-dimensionally arranged on a substrate and made of polylactic acid capable of perforating the skin, and the microneedles are coated with a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells) A method of increasing the response of Japanese encephalitis virus antigens using a device.
  12. 　前記マイクロニードルが円錐型または角錐型である、請求項１１に記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏功性を上昇させる方法。 The method for increasing the efficacy of a Japanese encephalitis virus antigen by the microneedle device according to claim 11, wherein the microneedle is conical or pyramidal.
  13. 　前記コーティングが、コーティング担体としてプルランを含む、請求項１１または１２に記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。 The method for increasing the response of Japanese encephalitis virus antigen by the microneedle device according to claim 11 or 12, wherein the coating contains pullulan as a coating carrier.
  14. 　前記コーティングが、室温での相対湿度７０．０～１００％ＲＨにて行われる、請求項１１から１３のいずれかに記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。 The method for increasing the response of Japanese encephalitis virus antigen by the microneedle device according to any one of claims 11 to 13, wherein the coating is performed at a relative humidity of 70.0 to 100% RH at room temperature.
  15. 　前記コーティングが、アジュバント活性を有する物質を含む、請求項１１から１４のいずれかに記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。 The method for increasing the response of Japanese encephalitis virus antigen by the microneedle device according to any one of claims 11 to 14, wherein the coating contains a substance having an adjuvant activity.
  16. 　前記アジュバント活性を有する物質が、ラウリルアルコールである、請求項１５に記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。 The method for increasing the efficacy of a Japanese encephalitis virus antigen by the microneedle device according to claim 15, wherein the substance having an adjuvant activity is lauryl alcohol.
  17. 　前記マイクロニードルデバイスの基板が、日本脳炎ウイルス抗原液または日本脳炎ウイルス抗原溶解液を伝達可能な複数の開口部を有するものである、請求項１１から１６のいずれかに記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。 The substrate of the microneedle device has a plurality of openings through which the Japanese encephalitis virus antigen solution or the Japanese encephalitis virus antigen solution can be transmitted. A method for increasing the response of encephalitis virus antigens.
  18. 　前記マイクロニードルの高さが２００～５００μｍである、請求項１１から１７のいずれかに記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。 The method for increasing the response of a Japanese encephalitis virus antigen by the microneedle device according to any one of claims 11 to 17, wherein the microneedle has a height of 200 to 500 µm.
  19. 　前記マイクロニードルが４００～１０００本／ｃｍ２の密度で配置される、請求項１１から１８のいずれかに記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。 The method for increasing the response of Japanese encephalitis virus antigen by the microneedle device according to any one of claims 11 to 18, wherein the microneedles are arranged at a density of 400 to 1000 / cm 2 .
  20. 　請求項１１から１９のいずれかに記載のマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法による日本脳炎ウイルス抗原の経皮投与後、更にアジュバント活性を有する貼付剤を貼付して、更なるマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。 After the transdermal administration of the Japanese encephalitis virus antigen by the method for increasing the response of the Japanese encephalitis virus antigen using the microneedle device according to any one of claims 11 to 19, a patch having an adjuvant activity is further applied. A method of increasing the response of Japanese encephalitis virus antigens using a microneedle device.
  21. 　前記貼付剤のアジュバント活性を有する物質が、ラウリルアルコールである、請求項２０に記載の更なるマイクロニードルデバイスによる日本脳炎ウイルス抗原の奏効性を上昇させる方法。 21. The method for increasing the response of Japanese encephalitis virus antigens with a further microneedle device according to claim 20, wherein the substance having the adjuvant activity of the patch is lauryl alcohol.
PCT/JP2009/062887 2008-07-30 2009-07-16 Microneedle device and method for increasing the response of japanese encephalitis virus antigen with the microneedle device WO2010013601A1 (en)

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KR20110036907A (en) 2011-04-12

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